In 2019, this project provides state-of-the-art research technologies that are developed, validated, and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the intramural program. Electron Microscopy The Electron Microscopy (EM) Unit provides expertise in advanced electron microscopy (EM)-related techniques and technologies to support the ultra-structural imaging needs of the DIR scientists both in Bethesda and at the Rocky Mountain Laboratories (RML). Real-time viewing of samples by video image sharing is available to all NIAID users, enabling scientists in Bethesda and Rockville to view samples simultaneously with the RML microscopy staff. The facility provides sample preparation, imaging, and analysis, ranging from basic structural studies and immune-localization of selected antigens to high resolution and three-dimensional analyses for a wide array of specimens. Optimal preservation of specimens is one of the major challenges in the field of EM, and the EM Unit uses a variety of methods, protocols, and equipment to accommodate different preparative and imaging needs. Conventional techniques for sample preparation inherently introduce structural changes in specimens, which can lead to artifacts. The EM Unit has put much effort into this critical first step as evidenced by the development of application-specific chemical and cryogenic preparative techniques to optimally preserve structures of interest for visualization and interpretation. These techniques in combination with new advanced imaging technologies and the ability to plunge freeze for near native state preservation, now allows visualization of macromolecular complexes, viruses, bacteria, and host-pathogen interactions at high resolution and in three dimensions. Concurrently, our bioinformatics component continues to evolve for high-resolution analyses of increasingly complex structures, and more efficient and comprehensive data collection for higher and better throughput. The broad educational background of the EM Unit staff, along with the opportunity to participate in microscopy meetings and hands-on workshops have been critical for expanding the repertoire of techniques. The Unit has expertise for both tomographic data collection and high resolution analysis tools, protocol development especially for immunology, optimizing parameters for techniques including data collection schemes, microwave-assisted freeze substitution (MWFS), and correlative light and electron microscopy (CLEM). Collaborations with various experts in structural biology and cryo-electron tomography (Cryo-ET) continue to provide both analytical assistance vital for determining viral and macromolecular structures, and automated tomographic alignments for improving database efficiency and workflow. New personnel include interns and contract hires who bring both microscopy and data management expertise. On-going developments in both techniques (i.e. immune-labeling, serial section SEM, montage tomography) and technologies (i.e. direct electron detector and the Volta phase plate) have expanded capabilities with significant improvement in 3D visualization and resolution potential at 3 angstroms. Collaborations resulting in improved data analysis, data management, specimen preparation and data collection have been invaluable for efficient developing highly specialized skills. Flow Cytometry Project-specific research applications are developed for flow cytometry analysis and sorting in BSL-2 and BSL-3 environments. RML Genomics Unit The RML Genomics Unit provides DIR investigators with expertise in experimental design, sample preparation, experimentation, and data analysis for high throughput genomic technologies such as Next Generation Sequencing (NGS), and qPCR. The RML Genomics Unit (GU) specializes in gene expression analysis of both host and/or pathogen, de novo assembly of bacterial genomes, and SNP/INDEL analysis of pathogen genomes and human exomes. The Unit also performs Sanger sequencing in a high throughput manner and assists investigators to troubleshoot difficult sequencing regions. The Unit's focus is to help the investigator optimize their use of genomic technologies. By using knowledge of the technologies and experience in experimental design and data analysis, the Unit can determine the ideal number of samples and replicates, the best technological platform, and the appropriate analysis to achieve the desired results in the most efficient manner. Visual and Medical Arts The Visual and Medical Arts Unit (VMA) at Rocky Mountain Laboratories (RML) provides highly specialized and advanced technologies to support the Division of Intramural Researchs (DIR) research program. The unit offers a wide variety of digital technologies, involving a comprehensive suite of illustration, design services, photography, 3D animation, video, and new media to document scientific findings. In addition, the VMA provided scientific communication to the Office of the Director, NIAID for presentation to scientists, the public as well as US lawmakers. The Visual and Medical Arts (VMA) Unit works in close collaboration with the research staff to provide the highest quality illustrations and images to assist in the visual communication of complex scientific ideas and findings. In addition to supporting research in the DIR, the VMA also provides varied media support for the National Institute of Allergy and Infectious Diseases (NIAID) Office of the Director. Some of these tasks have included creating visuals that were used in testimony to congress and foreign dignitaries.